Composition for in vivo transplantation for treatment of human cervical cancer comprising mononuclear cells derived from umblical cord blood

ABSTRACT

Provided is a composition for in vivo transplantation for the treatment of human cervical cancer, comprising mononuclear cells derived from umbilical cord blood and a pharmaceutically acceptable carrier. When the umbilical cord blood-derived mononuclear cells are transplanted in vivo, cervical cancer can be effectively treated. In particular, the mononuclear cells derived from the umbilical cord blood retain high differentiation and proliferation abilities and exhibit very low graft-versus-host (GVH) reactions which are side effects caused by transplantation, and thus, can be transplanted to many patients.

TECHNICAL FIELD

The present invention relates to a composition for in vivotransplantation for the treatment of human cervical cancer, comprisingmononuclear cells derived from umbilical cord blood.

BACKGROUND ART

Cancer is the second leading cause of death in humans. Chemotherapy(administration of anticancer drugs), radiation therapy, and surgeryhave been mainly used for cancer treatment. Cancer can be treated at theearly stage by using any one of the above methods or combination of themethods. However, for cancer which has already progressed to theterminal stage, which shows metastasis to other tissues through blood,or which shows a recurrence of cancer, the above methods have a very lowtherapeutic effect.

For most solid tumors, surgery is followed by chemotherapy and radiationtherapy. However, since the cancers cannot be completely eliminated, therisk of recurrence is remarkably increased due to residual cancer cells.Recent research has revealed that cancer stem cells are present invarious cancer tissues, and in order to fundamentally treat cancers, thecancer stem cells must be eliminated (Leukemia stem cells. Luo L, Han ZC. Int J Hematol. 2006. 84:123-127, Cancer stem cells—new andpotentially important targets for the therapy of oral squamous cellcarcinoma. Costea D E, Tsinkalovsky O, Vintermyr O K, Johannessen A C,Mackenzie I C. Oral Dis. 2006. 12:443-54. Cancer stem cells. Guo W,Lasky J L 3rd, Wu H. Pediatr Res. 2006. 59:59-64). However, markersspecific for cancer have not yet been found, which makes the fundamentaltreatment of cancer difficult.

In order to solve this problem, various cancer treatment techniques havebeen developed. Among them, attempts have been mostly made to treatcancers using cancer-specific antibodies or immune cells (A newgeneration of monoclonal and re-combinant antibodies againstcell-adherent prostate specific membrane antigen for diagnostic andtherapeutic targeting of prostate cancer. Elsasser-Beile U, Wolf P,Gierschner D, Buhler P, Schultze-Seemann W, Wetterauer U. Prostate.2006. 66:1359-70).

With respect to cancer treatment using cancer-specific antibodies,activation of immune cells and their complements using antibodiesagainst cell surface proteins which are expressed specifically in cancercells and administration of toxin-linked antibodies into human bodieshave been attempted.

With respect to cancer treatment using immune cells, techniques ofeliminating cancer cells by cancer cell-specific cytotoxic T cellsincluded in mass-cultured immune cells have been developed. That is,immune cells extracted from a blood sample provided by a cancer patientare mass-cultured in vitro and are then administered to cancer patients(Adoptive transfer of tumor-reactive Melan-A-specific CTL clones inmelanoma patients is followed by increased frequencies of additionalMelan-A-specific T cells. Vignard V, Lemercier B, Um A, Pandolfino M C,Guilloux Y, Khammari A, Rabu C, Echasserieau K, Lang F, Gougeon M L,Dreno B, Jotereau F, Labarriere N. J Immunol. 2005, 175:4797-805).

In addition, cell therapy using dendritic cells has been developed. Thisis a method of eliminating cancer cells by stimulating the activationand immune responses of T cells in vivo, the method including isolatingand culturing dendritic cells having an excellent antigen-presentingfunction that can present antigens specific to cancer cells, in vitro;adding a patient's cancer cell lysate or a cancer-specific antigenprotein to the culture solution; and administering the mixture to apatient (Vaccination of Japanese patients with advanced melanoma withpeptide, tumor lysate or both peptide and tumor lysate-pulsed mature,monocyte-derived dendritic cells. Nakai N, Asai J, Ueda E, Takenaka H,Katoh N, Kishimoto S. J Dermatol. 2006. 33:462-72. Clinical evaluationof dendritic cell vaccination for patients with recurrent glioma:results of a clinical phase I/II trial. Yamanaka R, Homma J, Yajima N,Tsuchiya N, Sano M, Kobayashi T, Yoshida S, Abe T, Narita M, TakahashiM, Tanaka R. Clin Cancer Res. 2005, 11:4160-7).

Recently, a method of treating cancer using a graft-versus-tumor (GVT)has been developed. According to this method, donor's blood sampleshaving genetically different human leukocyte antigens (HLAs) areextracted, and T cells and natural killer (NK) cells of the bloodsamples are administered to patients with cancer to inducegraft-versus-host (GVH) reactions, thereby eliminating residual cancercells (A phase 1 trial of donor lymphocyte infusions expanded andactivated ex vivo via CD3/CD28 costimulation. Porter D L, Levine B L,Bunin N, Stadtmauer F A, Luger S M, Goldstein S, Loren A, Phillips J,Nasta S, Perl A, Schuster S, Tsai D, Sohal A, Veloso E, Emerson S, JuneC H. Blood. 2006, 107:1325-31. Allogeneic hematopoietic stem celltransplantation for metastatic breast cancer. Bishop M R. Haematologica.2004, 89:599-605). This method can induce higher immune responses than amethod using autoimmune cells, and thus, is expected to be effective asa new cell therapy for the treatment of cancer.

Meanwhile, according to a conventional cancer treatment method usingimmune cells, i.e., a method of transplanting immune cells isolated fromperipheral blood, it is difficult to continue cancer treatment for along time due to the low proliferation ability of the transplantedimmune cells. Moreover, according to a cancer treatment method usingimmune cells isolated from peripheral blood, due to the GVH reactions,cells derived from a patient are cultured in vitro, and are thentransplanted to the patient. However, the cells cultured in vitro arecells in the terminal cell cycle stage, and thus, retain limited cellproliferation ability. Therefore, even though the cells are transplantedto a patient, it is difficult to expect the long-term therapeuticefficacy of immune cells.

DISCLOSURE OF INVENTION Technical Problem

The present invention provides for improvement in a conventional cancertreatment method for the treatment of cancer, especially cervicalcancer, using immune cells.

Technical Solution

When mononuclear cells derived from umbilical cord blood which can beisolated without significantly affecting human bodies were applied topatients with cervical cancer, it was found that the mononuclear cellsshow good regenerative potential of hematopoietic cells and that evenhuman leukocyte antigen (HLA)-mismatched patients exhibited very lowgraft-versus-host (GVH) reactions, thereby mononuclear cells derivedfrom umbilical cord blood being effective for the treatment of cervicalcancer.

Therefore, the present invention provides a composition for in vivotransplantation for the treatment of human cervical cancer, comprisingmononuclear cells derived from umbilical cord blood.

Advantageous Effects

According to the present invention, when mononuclear cells derived fromumbilical cord blood are transplanted in vivo, cervical cancer can beeffectively treated. In particular, the umbilical cord blood-derivedmononuclear cells retain high differentiation and proliferationabilities and exhibit very low graft-versus-host (GVH) reactions whichare side effects caused by transplantation, and thus, can betransplanted to many patients. That is, the umbilical cord blood-derivedmononuclear cells are differentiated and proliferated aftertransplantation, and thus, proliferation of cervical cancer cells iscontinuously prevented and immune rejection reactions in vivo hardlyoccur.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows flow cytometry results for stem cells and immune cells inmononuclear cells derived from umbilical cord blood (a shows CD3+ Tcells, b shows CD34+ hematopoietic stem cells, and c shows CD19+ Bcells);

FIG. 2 shows cervical tumors formed in mice transplanted with Caskicells, mice co-transplanted with Caski cells and umbilical cordblood-derived mononuclear cells, and mice transplanted with umbilicalcord blood-derived mononuclear cells 2-3 weeks after transplantationwith Caski cells;

FIG. 3 shows cervical tumors extracted from mice transplanted with Caskicells, mice co-transplanted with Caski cells and umbilical cordblood-derived mononuclear cells, and mice transplanted with umbilicalcord blood-derived mononuclear cells 2-3 weeks after transplantationwith Caski cells;

FIG. 4 shows sizes of tumors extracted from mice transplanted with Caskicells, mice co-transplanted with Caski cells and umbilical cordblood-derived mononuclear cells, and mice transplanted with umbilicalcord blood-derived mononuclear cells 2-3 weeks after transplantationwith Caski cells;

FIG. 5 shows weights of tumors extracted from mice transplanted withCaski cells, mice co-transplanted with Caski cells and umbilical cordblood-derived mononuclear cells, and mice transplanted with umbilicalcord blood-derived mononuclear cells 2-3 weeks after transplantationwith Caski cells; and

FIG. 6 shows umbilical cord blood-derived hematopoietic and immune cellspresent in the peripheral blood (a, b) and the spleen tissues (c) ofanimal models (mice transplanted with human cervical cancer cells).

BEST MODE FOR CARRYING OUT THE INVENTION

According to an aspect of the present invention, there is provided acomposition for in vivo transplantation for the treatment of humancervical cancer, comprising mononuclear cells derived from umbilicalcord blood and a pharmaceutically acceptable carrier. The umbilical cordblood-derived mononuclear cells may include CD34+ hematopoietic stemcells, CD3+ T cells and CD19+ B cells.

Mononuclear cells derived from umbilical cord blood according to thepresent invention are present as immature cells and retain remarkabledifferentiation and proliferation abilities. Cells obtained by in vitroculture of immune cells derived from peripheral blood are cells in theterminal cell cycle stage, and thus, retain limited cell proliferationabilities. Thus, even when the cells are transplanted to a patient, itis difficult to expect long-term cell therapeutic efficacy. However, theumbilical cord blood-derived mononuclear cells retain highdifferentiation and proliferation abilities and exhibit very lowgraft-versus-host (GVH) reactions which are side effects caused bytransplantation, and thus, can be transplanted to many patients. Thatis, the umbilical cord blood-derived mononuclear cells aredifferentiated and proliferated after transplantation, and thus, theproliferation of cervical cancer cells is continuously prevented andimmune rejection reactions in vivo hardly occur.

The umbilical cord blood-derived mononuclear cells can be isolated by aknown method, e.g., a modified Ficoll-Hypaque method, a 3% gelatinmethod, or a Ficoll-Hypaque method (Kim et al., Optimal umbilical cordblood processing: Basic study for the establishment of cord blood bank,Korean Journal of Hematopoietic Stem Cell Transplantation.2000.5:61-68). In addition, after adding an anticoagulant to umbilicalcord blood, mononuclear cells can be isolated by a Ficoll-Paque densitygradient centrifugation method.

The composition of the present invention can be administeredparenterally, e.g., in the form of subcutaneous injections. For example,the composition can be directly injected to cancer-forming tissue sites.For parenteral administration (e.g., in the form of injections), thecomposition of the present invention may be formulated into dispersionsand/or solutions including a pharmaceutically acceptable carrier, e.g.,sterilized deionized water, a buffer (about pH 7), or physiologicalsaline. If necessary, the composition of the present invention mayinclude an additive commonly used in the art, e.g., a preservative or astabilizer.

The composition of the present invention can be administered to adultpatients who suffer from cervical cancer at a dosage of 1×10⁷-10×10⁸cells/kg, preferably 2×10⁷-4×10⁷ cells/kg, more preferably about 2×10⁷cells/kg (based on an adult patient with an average weight of about 60kg). However, an adequate dosage can be changed according to the typeand conditions of a disease. For normal adult patients, the compositionof the present invention can be administered in a unit dosage formincluding about 2×10⁷ umbilical cord blood-derived mononuclear cells anda pharmaceutically acceptable carrier.

Hereinafter, the present invention will be described more specificallywith reference to the following examples. The following examples areonly for illustrative purposes and are not intended to limit the scopeof the invention.

EXAMPLES

Caski cells (Korean Cell Line Bank, Cat. NO. 21550), which were humancervical cancer cells, were injected into non-obese diabeticsevere-combined immunodeficient (NOD-SCID) mice to establish animalmodels for human cervical cancer. Mononuclear cells isolated fromumbilical cord blood were transplanted in vivo into the mice. The sizeand weight of tumors formed in the subcutaneous tissues of the mice weremeasured, and the presence of hematopoietic cells in the periphery bloodand the spleen of the mice in which the mononuclear cells weredetermined.

Example 1 Culture of Human Cervical Cancer Cells

Caski cells (Korean Cell Line Bank, Cat. NO. 21550), which were cellsderived from patients with cervical cancer, were cultured in RPMI(Rosewell Park Memorial Institute, Gibco-BRL, Korea) supplemented with10% fetal bovine serum (FBS, Jeil Biotech Services), 0.25 M HEPES(N-2-hydroxyethyl-piperazine-N′-2-ethane-sulfonic acid), and 1%penicillin and streptomycin.

Example 2 Isolation of Mononuclear Cells from Umbilical Cord Blood

Umbilical cord blood treated with an anticoagulant (heparin) was addedto a 50 ml Falcon tube containing 20 ml of a Ficoll-Paque solution(Amersham Biosciences AB, Sweden), and the mixture was then centrifugedat 2000 rpm at room temperature for 20 minutes. A mononuclear cellfraction of the middle layer was collected, diluted with a 2-fold volumeof a phosphate buffered saline (PBS), centrifuged at room temperaturefor five minutes for washing.

The mononuclear cells thus-obtained were stained with an anti-CD34antibody which is a stem cell-specific antibody, anti-CD3 and anti-CD 19antibodies which are immune cell-specific antibodies, and an anti-CD45antibody which is an antibody against CD45 which is expressed in wholemononuclear hematopoietic cells, for 30 minutes, and PBS (D-phosphatebuffered saline) was then added thereto. The mixture was centrifuged at1500 rpm for 5 minutes to remove antibodies that did not form anantibody-antigen complex. The mononuclear cells were washed and analyzedby a flow cytometer (FACSvantage, BD, U.S.A.) to determine the presenceof stem cells and immune cells in the mononuclear cells.

FIG. 1 shows flow cytometry results for stem cells and immune cells inthe mononuclear cells derived from the umbilical cord blood, a showsCD3+ T cells, b shows CD34+ hematopoietic stem cells, and c shows CD19+B cells. As shown in FIG. 1, the mononuclear cells from umbilical cordblood contained CD34+ hematopoietic stem cells, CD3+ T cells and CD19+ Bcells.

Example 3 Establishment of Experimental Animal Models and in vivoTransplantation of Mononuclear Cells Derived from Umbilical Cord Blood

NOD-SCID mice (6-8 weeks old) were divided into three groups of 5 miceeach.

For the first group, the Caski cells obtained in Example 1 (2×10⁶cells/mouse) in physiological saline were transplanted into thesubcutaneous tissues of the NOD-SCID mice using a 1 ml syringe. For thesecond group, the Caski cells obtained in Example 1 (2×10⁶ cells/mouse)in physiological saline were transplanted into the subcutaneous tissuesof the NOD-SCID mice using a 1 ml syringe, and incubated for abouttwo-three weeks to form cervical tumors. Then, the mononuclear cellsobtained in Example 2 (2×10⁷ cells/mouse) were transplanted into thetumor sites of the mice in the same manner as above. For the thirdgroup, the Caski cells (2×10⁶ cells/mouse) and the mononuclear cellsobtained in Example 2 (2×10⁷ cells/mouse) were transplanted into thesubcutaneous tissues of the NOD-SCID mice on the same day in the samemanner as above.

Example 4 Evaluation of Tumor Suppression Effect of Mononuclear CellsDerived from Umbilical Cord Blood

For the mice of each group treated according to Example 3, the size oftumors was measured using vernier calipers from 6 weeks after thetransplantation of the cervical cancer cells. At 8 weeks after thetransplantation, 100 μl of peripheral blood was extracted from each ofthe mice, and the presence of umbilical cord blood-derived T and B cellsand myeloid cells in the umbilical cord blood-transplanted mice wasanalyzed by flow cytometry. The mice were sacrificed, and tumors wereexcised and weighed (see FIG. 2 through 5).

FIG. 2 shows cervical tumors formed in the mice of the first throughthird groups, and FIG. 3 shows tumors extracted from the mice of thefirst through third groups. In FIGS. 2 and 3, a shows cervical tumors ofthe first group (the mice transplanted with only the Caski cells), bshows cervical tumors of the second group (the mice transplanted withthe umbilical cord blood-derived mononuclear cells 2-3 weeks after tumorinduction), and c shows cervical tumors of the third group (the miceco-transplanted with the cervical cancer cells and the mononuclearcells). FIGS. 4 and 5 shows a tumor size (FIG. 4) and a tumor weight(FIG. 5) in the mice of each group after 8 weeks.

Referring to FIG. 2 through 4, the mice co-transplanted with thecervical cancer cells and the umbilical cord blood-derived mononuclearcells exhibited a significantly higher tumor suppression effect comparedto the mice transplanted with only the cervical cancer cells. The micetransplanted with the umbilical cord blood-derived mononuclear cells twoweeks after tumor induction also exhibited a tumor suppression effect(P<0.05). Referring to FIG. 5, a tumor suppression effect (0.02±0.02) ofthe mice co-transplanted with the cervical cancer cells and theumbilical cord blood-derived mononuclear cells was much higher than that(1.7±0.24) of the mice transplanted with only the cervical cancer cells.The mice transplanted with the umbilical cord blood-derived mononuclearcells 2-3 weeks after tumor induction also exhibited a tumor suppressioneffect (0.7±0.24) (P<0.05).

Example 5 Determination of Presence of Umbilical Cord Blood-DerivedCells in Mice

In order to determine the presence of umbilical cord blood-derivedmononuclear cells in the mice whose the subcutaneous tissues weretransplanted with the umbilical cord blood-derived mononuclear cells(the second and third groups), 500 μl of the peripheral blood extractedfrom the mice before sacrificed was placed in ananti-coagulant-containing tube, and mononuclear cells were isolated fromthe peripheral blood using a Ficoll-Paque solution (Amersham BiosciencesAB, Sweden) in the same manner as in Example 2. On the other hand,mononuclear cells were isolated from the spleen tissues of the micewhose the subcutaneous tissues were transplanted with the umbilical cordblood-derived mononuclear cells (the second and third groups) using aFicoll-Paque solution (Amersham Biosciences AB, Sweden) in the samemanner as in Example 2.

The mononuclear cells were stained with an anti-human CD45 antibodywhich is a marker specific to human mononuclear hematopoietic cells, andwith antibodies specific to human immune cells (B cells, NK cells, and Tcells), and the presence of human umbilical cord blood-derivedhematopoietic and immune cells in animal models was determined by flowcytometry (see FIG. 6).

FIG. 6 shows umbilical cord blood-derived hematopoietic and immune cellspresent in the peripheral blood (a, b) and the spleen tissues (c) of theanimal models (the mice transplanted with the human cervical cancercells). Referring to FIG. 6, the CD45+ cells, which are humanhematopoietic cells, and the CD3+ cells, which are human T cells, areobserved in the mononuclear cells derived from the peripheral blood (a,b), and the CD4+ cells, which are human umbilical cord blood-derivedhelper T cells, and the CD8+ cells, which are cytotoxic T cells, areobserved in the mononuclear cells derived from the spleen tissues.

1-2. (canceled)
 3. A method for treating a human cervical cancer,comprising transplanting to a subject in need a composition comprisingmononuclear cells derived from umbilical cord blood and apharmaceutically acceptable carrier.
 4. The method of claim 3, whereinthe mononuclear cells derived from the umbilical cord blood compriseCD34+ hematopoietic stem cells, CD3+ T cells and CD19+ B cells.